Rev. bras. farmacogn. vol.25 número5

RevistaBrasileiradeFarmacognosia25(2015)529–532

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Short

communication

Microsomal

metabolism

of

erythraline:

an

anxiolitic

spiroalkaloid

Lucas

Maciel

Mauriz

Marques

_,

_Fernando

_Armani

_Aguiar

_,

_Denise

_Brentan

_da

_Silva

_,

Daniel

Roberto

Callejon

_,

_Anderson

_Rodrigo

_Moraes

_de

_Oliveira

_,

_Norberto

_Peporine

_Lopes

_,

João

Luís

Callegari

Lopes

_,

_Thais

_Guaratini

a_{NúcleodePesquisaemProdutosNaturaiseSintéticos,FaculdadedeCiênciasFarmacêuticasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil} b_{LychnofloraPesquisaeDesenvolvimentoemProdutosNaturaisLtda,CampusUSP,RibeirãoPreto,SP,Brazil}

c_{DepartamentodeQuímica,FaculdadedeFilosofia,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,Brazil}

a

r

t

i

c

l

e

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n

f

o

Articlehistory:

Received25September2014 Accepted4May2015 Availableonline8July2015

Keywords:

Fabaceae Erythraline CYP450 Drugmetabolism Microsomalmodel

a

b

s

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c

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ThegenusErythrina,Fabaceae,iswidelydistributedintropicalandsubtropicalregions.Theirflowers, fruits,seedsandbarkarefrequentlyusedinfolkmedicineforitseffectsonthecentralnervoussystem suchasanticonvulsant,antidepressant,analgesic,sedative,andhypnoticeffects.Erythralinehasbeen reportedasoneoftheactivecompoundsfromErythrina,butuntilnowtherearenopharmacokinetics dataaboutthiscompoundandonlyfewresultsshowingaputativemetabolismwerereported.Toimprove theinformationabouterythralinemetabolism,thisarticlereportsanddiscusses,forthefirsttime,the

invitrometabolismbiotransformationoferythralinebycytochromeP450enzymes.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Naturalproductshavehistoricallyplayedamajorroleinthe dis-coveryanddevelopmentofadiversearrayoftherapeutics(Kurita and Linington,2015).Theyrepresent a rich sourceof chemical diversity,between25and50%ofapproveddrugsareoriginated fromnaturalproducts.However,discovery,isolation,and develop-mentofnaturalproductsaspharmaceuticaldrugsareexceptionally challenging,requiringamulti-disciplinaryapproachtounlocktheir potential(OgbourneandParsons,2014).

ThegenusErythrina,Fabaceae,iswidelydistributedintropical andsubtropicalregions(Aritaetal.,2014).ErythrinavernaVell.isa deciduoustreefoundmainlyinBraziliancerradosandother trop-icalregions.Itsflowers,fruits,seedsandbarkarefrequentlyused infolkmedicineduetotheireffectsonthecentralnervous sys-tem,suchas:anticonvulsant,antidepressant,analgesic,sedative andhypnoticeffects(Faggionetal.,2011;Flausinoetal.,2007a,b). Systematicphytochemicalinvestigationsreportedtheoccurrence ofoveronehundredstructuralErythrinaalkaloidsderivatives(Juma andMajinda,2004;Wanjalaetal.,2002).Biologicalstudies regard-ingitsanxiolyticpropertieshavesupportedthepopularuseand themechanismofactionwasdescribedas␣4␤2nicotinic recep-torsantagonist.Inthelastdecade,initialpharmaceuticalproducts

∗ Correspondingauthors.

E-mails:joaoluis@usp.br(J.L.C.Lopes),thais@lychnoflora.com.br(T.Guaratini).

werecommerciallyavailable,butuntilnowthereareno pharma-cokineticsdataoftheactivecompoundsandonlyfewresultsabout putativemetabolismhavebeenreported(Guaratinietal.,2014; Perdigaoetal.,2013).

Metabolism of a drugcandidate often decides whetherit is safeand effective for clinicalapplication.The understandingof specificdrugmetabolitesisoftenakeyforthesuccessofdrug dis-covery(Cusacketal.,2013;Chenetal.,2014)anditiscrucialto informseveralimportantstructuralpropertiesasnew pharmaco-logicallyinactiveortoxicentities,poorbioavailability,efficacy,and safety-relatedevents.Therefore,suchanearlyscreeningindrug developmentisnecessary(Fragaetal.,2011).

Ratand humanlivermicrosomes(RLMandHLM)aremainly employedformetaboliteprofilingpurposes(Spaggiarietal.,2014; Yuanetal.,2014).Thesystemcontainsthemaindrug-metabolizing enzymes,suchasthecytochromeP450(CYP450)familyandflavin monooxygenase(Chenetal.,2014),PhaseIandPhaseIIenzymes, whichcangeneratevaluableinformationtopredictandavoid prob-lemsduringinvivostudies(Nowaketal.,2014).

Inthisway,ourgrouphasbeendedicatedtoinvestigatePhase I metabolites for a series of natural products (Marques et al., 2014; Messiano et al., 2013; Moreiraet al., 2013).Analysis of erythraline (1, ERT) metabolization by pig microbiota suggests a high stability in intestinal portion, but applying bioorganic catalysis,oneputativemetabolitewasformedand identifiedas 8-oxo-erythraline (2) (Guaratini et al., 2014). Considering ERT (1)asa promising drugcandidate,itsmetabolismstill requires

http://dx.doi.org/10.1016/j.bjp.2015.05.011

530 L.M.M.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)529–532

clarifications. Therefore, the aim of the present work was to investigateitsinvitrometabolismbylivermicrosomesobtained fromrats,aswellasfromhumanbeings,inordertopredictthe invivometabolism,comparingwithpreviousrelatedresultsand toimprove pre-clinical information of alkaloids from Erythrina genus.

Materialsandmethods

TheERT(1)wasextractedfromErythrinavernaVell.,Fabaceae, accordingtopublishedprocedures(Guaratinietal.,2014)andsome controlanalogswereobtainedaspreviouslydescribed(Callejon etal.,2014).Sodiumchlorideandsodiumdihydrogenphosphate wereobtainedfromMerck(Darmstadt,Germany).Sodium hydrox-ide and potassium chloride were obtained from Nuclear (São Paulo,Brazil).Glycerolandtris-(hydroxymethyl)-aminomethane wereobtainedfromJ.T.Baker (Phillipsburg,NJ, USA), ethylene-diaminetetraacetic acid (EDTA) from Carlo Erba (Milan, Italy). NADP+_{,glucose-6-phosphateandglucose-6-phosphate}

dehydro-genasewereobtainedfromSigma–Aldrich(St.Louis,MO,USA). Male Wistar rats weighing 180–220g were obtained from the School of Pharmaceutical Sciences of Ribeirão Preto, Uni-versity of Sao Paulo. The Ethical Committee from University of São Paulo approved the studies. Microsomal preparation wasperformedaccordingpublishedprocedures(Marquesetal., 2014). Ratliver microsome incubations(shaking water bath at 37◦_{C) wereperformed in10mlamber tubes in atotal} incuba-tion volume of 1000␮l. Incubations contained 250␮l cofactor solution,35␮lratlivermicrosomes(29.6mgml−1_microsomal pro-tein), 715␮l phosphate buffer (pH 7.4; 0.25moll−1_{) and 25␮l} ERT(1;500␮gml−1_{).The cofactor solutionconsistedof NADP}+ (0.25mmoll−1_{),glucose-6-phosphate(5mmoll}−1_)and glucose-6-phosphatedehydrogenase(0.5units)inTris–HClbuffer(Tris–HCl 0.05moll−1_{–KCl0.15moll}−1_,pH7.4).

Humanlivermicrosomeincubations(BDGentestTM_,Woburn,

MA,EUA)wereperformedaccordingClementsandLi(2011):to avoidmultiplefreeze–thawcyclesandmaintainenzymeactivity themicrosomesand NADPHregenerating system weredivided intosingleusefractionsandstoredat−94◦Cuntilneeded.Amber

tubesinatotalincubationvolumeof200␮lwereused.Incubations contained100␮lhumanlivermicrosome(4mgml−1_microsomal protein),100␮lNADPHregenerating system(NRS)(40␮l0.5M phosphatebufferpH7.4,10␮lSolutionA,2␮lSolutionB, both fromBDGentestTM_{),5␮lERT(1;1.38mgml}−1_).

Afterprewarmingfor5minat37◦_{C, themetabolicreactions} wereinitiatedbytheadditionoftheratandhumanliver micro-somes,separately.Tostopmetabolicreactionafter90min,itwas added4mland1mlofchloroform,respectively,toincubationswith ratandhumanlivermicrosomes.Then,thesamplepreparationwas performed.Controlincubationswereperformedintheabsenceof thecofactorsolutionandintheabsenceofthemicrosomal prepa-ration.Thedifferencebetween‘with’and ‘without’NADPHwas consideredCYP450-mediatedmetabolism.Toperformthe experi-ments,thesampleswerepooledfromtensingleincubations.

Aliquid–liquidextraction(LLE)procedurewasappliedtoextract theERT(1)fromtheratandhumanlivermicrosomes.Afterthe extractionprocedure,thesampleswereshakenfor15min(Vibrax

VXR agitator,IKA,Staufen, Germany)and centrifugedfor 5min at2860×g (HitachiCF16RXII,Himac,Tokyo,Japan). The

super-natantwascollected(3mland750␮l,respectively,fromratand humanmicrosomalpreparationsamples)andallowedto evapo-ratetodrynessunderagentlestreamofnitrogen.Then,theresidue wasreconstitutedin200␮lofmethanol,and1␮lwasinjectedinto thechromatographysystem.

In order to investigate the formation of metabolites, the samples were analyzed by a gas chromatograph (GC-MS-QP-2010, Shimadzu) coupled to a quadruple mass spectrometer with electron impact (EI) ionization at 70eV. The gas chro-matograph was equipped with an auto sampler AOC-20i, 1␮l splitinjections1/10wereperformedat250◦_{C.Separationswere} carried out ona 5% phenyl methyl siloxane (DB-5ms) column (30m×0.25mm×0.25␮mfilmthickness).Theoventemperature

program for separation conditions were 100◦_{C for 0.8min} fol-lowedbytemperatureincreasesto220◦_Cat6◦_Cmin−1_,220◦_Cfor 10min,temperatureincreasesto290◦_Cat6◦_Cmin−1_,and290◦_C for11min.Thecarriergaswasultrapureheliumhelium(grade5.5) atconstantflowratesof1.10mlmin−1_{.Amassrangeofm/z50–500} wasrecordedinthefull-scanmode.Finally,themetaboliteswere comparedwithauthenticallysamplespreviousisolated.

Toaccumulateenoughmetaboliteusedfor1_{HNMRanalysis,rat}

livermicrosomeincubationwasperformedaspreviousdescribed to150individualtubes(ERT,1,2000␮gml−1_{).Then,thispoolwas} submittedtosemipreparativeLCusingthefollowingconditions: C18 column (Shim-pack Prep-ODS, 5␮m, 20mm×25cm,

Shi-madzu),flowrate9mlmin−1_{andacetonitrile(B)andH2O(A)both} withTFA0.02%(v/v)assolvents.Theelutionprofilewas0–25min (15–65%,B),25–30min(25–80%,B),30–32min(80–100%,B)and 32–35min(100%,B).The isolatedcompound wascharacterized by NMR (Bruker Avance® _{DRX-500) in a Shigemi symmetrical}

susceptibility-matchedmicrotube.

Resultsanddiscussion

PureERT(1)elutes inGC–MSanalysisatRt 30.6min(Fig.1, charts A and B) and no other signal was observed. The chro-matogram of thetest reaction employing rat and humanliver microsomespresentedanewpeakatthesameRt29.81min show-ingthattherewasacorrelationbetweenbothmicrosomalmodels (Fig.1,chartsAandB).Theabsenceofsignalsintheblankand con-trolsamplesconfirmtheenzymaticconversionprobablythought anoxidativereaction.Theelectronionizationmassspectraofthe signal1atRt 29.81showanincrementof14massunits(Fig.1, chartD),relatedtoERT(Fig.1,chartC).Thisresultsuggestsan oxi-dationthroughacarbonylgroupformationaspreviousobserved forothernaturalproducts(Niehuesetal.,2012;Santosetal.,2005, 2008).Thefragmentationpatternofbothcompoundsshowed simi-larneutraleliminationsconfirmingthehomologybetweenthetwo structures.

Toconfirmthemetabolitestructurelarge-scaleratliver micro-someincubation wasperformedaspreviousdescribed (applied 150individualtubesofERTat2000␮gml−1_{).Then,thispoolwas} submittedtosemipreparativeLCaffording900␮g.Afterthe sol-ventelimination,thesamplewascharacterizedbyNMR(Bruker Avance® _{DRX-500) in a Shigemi symmetrical}

susceptibility-matchedmicrotube.The1_HNMR(CDCl

3,500MHz)spectraofthe

isolatedmetaboliteexhibitedthesignals(seesupplementarydata associatedwiththisarticle):ıH6.33(m,H-1),6.72(s,H-14),6.72

(s,H-17),6.89(m,H-2),6.15(s,H-7),5.94(d,O-CH2-O),5.91(d,

O-CH2-O),3.90(m,H-3),3.78(m,H-10),3.68m(m,H-10),3.35(s,

OCH3),3.15(m,H-11),2.99(m,H-11),2.82(m,H-4),2.10(m,H-4).

L.M.M.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)529–532 531

30,0

100 90 80 70 60 50 40 30 20 10

40

51 65

77 91 ₁₀₃

112 ₁₃₀ 152 180

191 208

213

226 239

264 266

282 297

60 80 100 120 140 160 180 200 220 240 260 280 300

100 90 80 70 60 50 40 30 20 10

51 ₆₃ 77 89 105

116 139 ₁₅₂

165

176 194

196

222

250

252

280 278

296 311

40 60 80 100 120 140 160 180190 210 230 250 270 290 310 m/z m/z T

1 2

1,000,000 1,000,000

2

1

T

C

B

TIC TIC

A

B

C

D

C B

31,0 32,0 33,0 34,0

mm 30,0 31,0 32,0 33,0 mm34,0

Fig.1. RepresentativeGCchromatogramsofrat(chartA)andhumanmicrosomesextracts(chartB),signal1:8-oxo-erythraline(Rt=29.81min)andsignal2:ERT

(Rt=30.6min).LinesT(testsamples)ofERTmetabolismafterincubation;linesC(control)inbothmicrosomalpreparationwithoutNADPHcofactor;linesB(blank):

microsomalpreparationwithoutERT.Left:ratlivermicrosomes;right:humanlivermicrosomes.ChartCshowstheEI-MSdataofERT(Rt=30.6min)andchartDshowsthe

EI-MSdataof8-oxo-erythraline(Rt=29.81min).

ionmode)confirmedthemolecularstructure(observ.m/z312.1228 [M+H]+_;calcd.forC

18H18NO4+312.1230,error=0.6ppm).

Conclusion

Thispaperdescribes,forthefirsttime,theCYP450-mediated metabolismofapromisingnaturalproduct,ERT(1),usingratand humanlivermicrosomes.Itsmetabolismshowedtheformationof thepreviouslyformed8-oxo-erythraline(2)bybioinorganic cataly-sis(Guaratinietal.,2014).Thiscompoundwaspreviouslyisolated asaminorcompoundinsomecultivarofE.vernaandbiological evaluationapplyingmacrophageandLeishmaniacellsrevealedlow cytotoxicactivityfortheERTanalogalkaloidsinvestigated.Inthis manner,thiscompoundshouldbeconsideredaspossible metabo-liteintheinvivometabolismanditstoxicologicaleffectmustbe furtherinvestigated.

Authors’contributions

LMMM,FAA,TG,DBS,DRCconductedextractionandisolation oftheeritralinandmetabolitesfromplantandinmicrosomal bio-transformation, respectively and the interpretation of all these data. LMMM, ARMO, NPL, JLCL and TG wrote and revised the manuscript.Allauthorsreadandapprovedthefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

532 L.M.M.Marquesetal./RevistaBrasileiradeFarmacognosia25(2015)529–532

CNPq(168114/2014-3),INCTifandRHAEforfellowshipsand finan-cialsupport.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.05.011.

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